CN106754748B

CN106754748B - Aedes albopictus densovirus and application thereof

Info

Publication number: CN106754748B
Application number: CN201611149407.6A
Authority: CN
Inventors: 顾金保; 刘培文; 陈晓光
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-12-14
Filing date: 2016-12-14
Publication date: 2020-08-18
Anticipated expiration: 2036-12-14
Also published as: CN106754748A

Abstract

The invention discloses an Aedes albopictus densovirus, which is named as Aedes albopictus densovirus AalDV-6 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO. V201652. Also discloses the application of the Aedes albopictus densovirus in the preparation of biological mosquito killing agents.

Description

Aedes albopictus densovirus and application thereof

Technical Field

The invention belongs to the technical field of mosquito densonucleosis viruses, and particularly relates to Aedes albopictus densovirus and application thereof.

Background

Mosquito-borne infectious diseases are diseases transmitted by taking mosquitoes as vectors, and comprise malaria, filariasis, dengue fever, epidemic encephalitis B, yellow fever and the like. The mosquito-borne diseases have wide epidemic range, strong transmission capacity and high incidence rate, and the mosquito-borne diseases are still prosperous all over the world despite the efforts of controlling for centuries. Worldwide, millions of people become infected with malaria each year, which causes the death of over 100 million children each year, mainly in sub-saharan africa. The main transmission vectors of dengue are aedes albopictus and aedes aegypti, which have been growing in the past decades as their transmission vectors have spread globally, returning to the area where they were eliminated in the mid-20 th century and causing a widespread hemorrhagic fever, and the aedes albopictus originated in southeast asia. West nile virus has become an epidemic throughout america over the last 10 years, while chikungunya virus has emerged in the indian basin and asian continents, affecting millions of people. Japanese encephalitis virus is also sub-spread in indian subcontinent and australia, mainly affecting young children. In China, 18 genera, 371 species and subspecies of mosquitoes are known, and main vectors of malaria, dengue fever, Japanese encephalitis and filariasis are covered. The epidemic of mosquito-borne infectious diseases seriously harms the life health of human beings, influences the stability of society and hinders the development of economy. Efforts to limit the impact of mosquito diseases in endemic areas face the dual challenges of controlling mosquito populations and providing effective public health interventions.

Vector mosquito control is an important measure for controlling the epidemic of mosquito-borne infectious diseases. At present, no specific treatment method or specific prevention means for mosquito-borne viral infectious diseases such as dengue fever, yellow fever and west nile virus disease exist, the importance of vector control in preventing the occurrence and prevalence of the virus is more prominent, and the search for an effective control method is an important subject facing the world medical community. The medium chemical control has a dominating position in mosquito medium control due to its outstanding effect, but along with the serious environmental pollution caused by the traditional chemical control, the disadvantages of easily causing drug resistance of mosquitoes become increasingly apparent, and the biological control of mosquitoes is more and more favored by people. The biological control mainly uses mosquito pathogens, parasites and predators to achieve the control effect, is harmless to non-target organisms and beneficial organisms, does not pollute the environment, and is not easy to generate resistance to mosquitoes.

The Mosquito Densovirus (MDV) belongs to the genus of densovirus of the family of parvoviridae (Parvovirinae), is a single plus-strand DNA virus, has a virus capsid without coating and has a plus-icosahedral structure with a diameter of 20 nm. The genome of the mosquito densovirus is about 4000nt long and consists of coding regions of left non-structural proteins NS1 and NS2, coding regions of right structural protein VP, and repetitive sequences (IRS) at both ends. The nonstructural protein NS1 mainly assists virus replication and activates transcription of structural proteins. The structural protein VP is the capsid protein IRS of the virus and serves as the viral replication initiation site and the packaging signal.

MDV is a pathogen that specifically infects mosquitoes, causing the death or morbidity of the host when the nuclear dense proliferation disease (densonucleosis) characteristic of the host is severe. The virus is sequentially separated from various mosquitoes such as Aedes aegypti, Aedes albopictus, anopheles minutissima and the like in nature and various laboratory mosquito cell lines/laboratory strains, and at present, 20 strains are obtained by co-separation, and about 60 percent of the virus is obtained by field population separation. The host specificity is strong, only mosquitoes are infected, and experiments prove that the mosquito-repellent incense cannot infect other kinds of insects, fishes, birds and mammals including human beings.

MDV has lethal effect on Aedes albopictus individuals under high titer condition, and has sublethal effect on mosquito individuals under low concentration condition, including prolonging larval stage of Aedes albopictus larvae, prolonging pupation time, prolonging eclosion time, etc. The virus in the mosquito breeding water can invade various tissues and organs such as mosquito fat, adult discs, cells around the trachea, Marsdenia tenacissima and the like. After the mosquito larvae are infected with MDV, the activity is greatly reduced, the mosquito larvae are distorted, and after the mosquito larvae are stimulated, the mosquito larvae have spastic convulsion and die partially. The surviving larva has delayed pupation and eclosion time and is easy to die when molting. The life of adult mosquitoes infected by virus is shortened, so that the opportunity of transmitting pathogen by the mosquitoes is reduced, and the egg laying rate and the egg laying activity of infected female mosquitoes are greatly reduced. The virus can be horizontally transmitted by larvae and adults and also can be vertically transmitted by female mosquitoes, so that the virus can be spread to various breeding water bodies and continuously continue in the natural world.

The pathogenic characteristics of the mosquito densovirus MDV such as host specificity, biological safety and the like enable the mosquito densovirus MDV to have the advantages that the potential value of mosquito control is incomparable with other types of pathogens, have the application prospect of being used as a high-efficiency mosquito biological insecticide and have the potential value in the aspect of mosquito-borne infectious disease control.

Disclosure of Invention

The invention aims to solve the technical problem of providing Aedes albopictus densovirus, a preparation method thereof and application thereof in preparing biological mosquito killing agents.

The technical problem of the invention is realized by the following technical scheme: an Aedes albopictus densovirus, named AalDV-6, is preserved in China center for type culture Collection with the preservation number of CCTCCNO.V201652.

The virus Aedes albopictus densovirus AalDV-6 is obtained by separating Aedes albopictus captured in the open air of Guangzhou for the first time by professor Gujinbao of southern medical university, and deeply researches the aspects of in-vitro cell culture, morphological structure, safety and the like, wherein the virus genome structure is shown in figure 4.

The invention also provides the complete sequence of the Aedes albopictus densovirus AalDV-6, the nucleotide sequence of which is shown as the sequence table SEQ ID NO.1, the sequence comprises an inverted repeat sequence ITR of the Aedes albopictus densovirus, two non-structural protein coding sequences NS1 and NS2, and a structural protein coding sequence VP.

The preparation method of the Aedes albopictus densovirus AalDV-6 comprises the steps of culturing the cell line multiplication mosquito densovirus AalDV-6 strain and feeding mosquito larvae multiplication mosquito densovirus AalDV-6. The method for culturing and proliferating the mosquito densovirus by feeding a large amount of mosquito larvae can greatly improve the virus yield, reduce the virus production cost and is relatively simple and convenient to operate.

Specifically, the method for culturing and proliferating the mosquito cell line comprises the following steps:

(1) inoculating Aedes albopictus densovirus AalDV-6 (obtained by separation in the invention) to an susceptible mosquito cell line, culturing the inoculated cells, and harvesting a cell culture;

(2) and (2) repeatedly freezing and thawing the cell culture obtained in the step (1) for multiple times, centrifuging, and collecting supernatant, namely the suspension of the mosquito densovirus AalDV-6.

In step (2), the cell culture in step (1) is preferably repeatedly frozen and thawed 3 times, and the centrifugation is preferably 3750 rpm for 15 minutes.

The susceptible cells can be continuous cell lines or primary cells. Susceptible cells suitable for mosquito densovirus include, but are not limited to, the C6/36 cell line of Aedes albopictus (ATCC accession No. CRL-1660), the Aedes aegypti aag2 cell and other continuous cell lines; primary cells can be isolated and prepared from mosquito larvae or tissues thereof by methods well known in the art.

The susceptible cell is suitable for infection and proliferation of mosquito densovirus. Including but not limited to mosquito-derived cell lines such as Aedes albopictus C6/36 cells, Aedes aegypti aag2 cells, and the like.

The method for preparing the mosquito densovirus by feeding the larvae comprises the following steps:

(1) each group exposed about 200 susceptible mosquitoes to 10⁸In the mosquito densovirus AalDV-6 (from the mosquito densovirus AalDV-6 suspension) per ml, feeding infected mosquito larvae, collecting dead larvae, and collecting all larvae after 7 days;

(2) grinding the larvae obtained in the step (1) with 1ml of PBS, centrifuging at 3750 rpm for 15 minutes, filtering and sterilizing the supernatant with a 0.22 mu m filter membrane, wherein the filtrate is the mosquito densovirus suspension.

The susceptible mosquito is a susceptible mosquito suitable for infecting mosquito densovirus. Including but not limited to mosquito larvae such as Aedes albopictus, Aedes aegypti, Culex fatigae, etc.

Further, the preparation method of the Aedes albopictus densovirus AalDV-6 comprises the following steps:

(1) viral infection of larvae

Putting the molting aedes albopictus larva into a mosquito densovirus AalDV-6 (obtained by separation of the invention), feeding the larva with turtle food after 24 hours, and gradually proliferating the larva in vivo after the virus infects the larva;

(2) virus isolation

Collecting dead larva bodies every day in the feeding process, storing at low temperature, grinding all the larvae with PBS, centrifuging, filtering and sterilizing supernate with a 0.22 mu m filter membrane, sucking 50 mu L of extracted DNA, quantifying viruses by adopting real-time fluorescent quantitative PCR, and storing the rest at low temperature to obtain suspension containing the mosquito densovirus AalDV-6.

The breeding conditions in the steps (1) to (2) are preferably constant temperature of 28 +/-1 ℃, humidity of 70-80% and light and dark in each day for 12 hours.

Preferably, about 200 newly molted aedes albopictus larvae are added into 200ml 10 ml in step (1)⁸In the mosquito densovirus AalDV-6 at a concentration of one virus per milliliter.

After a preferred 7-day period in step (2), all larvae are triturated with 1ml PBS, centrifuged at 3750 rpm for 15 minutes and the supernatant is sterile filtered through a 0.22 μm filter.

The virus concentration quantification method may be performed by a real-time quantitative fluorescent PCR absolute quantification method known in the art, such as the SYBR Green method described in example 2 or the Taqman probe method. The method of example 2 uses the following primers:

a forward primer: 5'-CAGGAGGAAACAGCACAAGA-3' the flow of the air in the air conditioner,

reverse primer: 5'-GTTTCGATACCGTAACGGATGC-3' the flow of the air in the air conditioner,

the annealing temperature is 55 ℃, and the fluorescent quantitative PCR reaction system is prepared as follows:

2 SuperReal Green PreMix Plus	10μL
		form panel	1μL
Forward primer (10 μm)	0.6μL
		Reverse primer (10 μm)	0.6μL
50 ROX Reference Dye	0.4μL
		RNase-free	7.4μL

The fluorescent quantitative PCR instrument adopts ABI 7500 of life company, and the reaction program is as follows:

the use of Aedes albopictus densovirus in the preparation of a biological mosquito repellent.

The invention also provides a biological mosquito killer which comprises Aedes albopictus densovirus AalDV-6 and is used for exposing Aedes albopictus larvae to 10¹⁰The mosquito of Aedes albopictus with half lethal time LT50 of AallDV-6 per ml is 11.54 days.

The invention has the following advantages:

(1) the Aedes albopictus densovirus provided by the invention is a virus only infecting mosquitoes, but not infecting other insects, fishes, birds and mammals including human beings, and the virus is highly parasitic and completely depends on the energy and metabolic system of host cells to obtain substances and energy required by life activities.

(2) The Aedes albopictus densovirus in the invention leaves a host cell, is a large chemical molecule, stops moving, can be prepared into protein crystals, is a non-living body, shows typical living body characteristics through adsorbing, entering, replicating, assembling and releasing progeny viruses only when meeting mosquitoes and cells thereof, shows safety according to the characteristics, has lower requirements on conditions such as temperature, time and the like during storage and transportation compared with other species of biological mosquito killers developed by bacterial fungi, and has lower transportation and storage cost.

(3) Along with the prevalence of mosquito-borne infectious viruses such as dengue fever, Zika fever, chikungunya fever and the like, the problem of the resistance enhancement of mosquitoes to chemical insecticides is gradually highlighted, the invention of a novel mosquito-borne control method is particularly important, and the mosquito densovirus can be developed into a high-efficiency and safe biological mosquito killer.

Drawings

FIG. 1 shows the proliferation of C6/36 cells within 67 days of the infection with the mosquito densovirus AalDV in example 2;

FIG. 2 shows the proliferation of the mosquito densovirus AalDV-6 in the Aedes albopictus larvae and in the breeding water in example 3;

FIG. 3 is an electron micrograph of the mosquito densovirus AalDV-6 purified after the culture of C6/36 cells in example 4;

FIG. 4 is the mosquito densovirus AalDV-6 genome of example 5, in which AalDV-6 genome is 3961nt in full length, encodes two nonstructural proteins NS1 and NS2, and a structural protein VP, and the viral genome is flanked by Inverted Terminal Repeat Sequences (ITRs);

FIG. 5 shows that in example 6, the mosquito densovirus AalDV-6 was at 10¹⁰Cumulative mortality of aedes albopictus at the dose of individual virus/ml.

Detailed Description

The present invention is specifically illustrated in the following examples. These examples are illustrative and are not intended to limit the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Defining:

"coding sequence" in the present application refers to a DNA sequence which can be transcribed to give the corresponding RNA sequence.

"ITR" as used herein refers to inverted terminal repeats flanking the viral genome, and is one of the characteristics of the parvoviridae family, having an ITR structure that has multiple functions of regulating viral gene replication, transcription, viral rescue, and the like.

"NS 1 and NS 2" in this application refer to viral

nonstructural proteins

1 and 2, which primarily function to aid in viral transcription, packaging, and the like.

"VP" in this application refers to a viral structural protein, the structure of which primarily expresses the capsid of the virus.

"vector" in this application refers to an artificially constructed recombinant plasmid for cloning.

"AalDV-6" in this application refers to a mosquito densonurus separated from Aedes albopictus, since it is reported worldwide that 5 strains of mosquito densonurus are separated in Aedes albopictus or in the C6/36 cell line of Aedes albopictus, the virus strain separated in this invention is the sixth strain separated from Aedes albopictus, and is named AalDV-6 according to the virus classification naming rule issued by International Committee for Committee of Committee on Taxonomy of Viruses, ICTV 2015, and the preservation number of this virus in the China center for typical microorganisms is CCTCC V201652.

"LT 50" in this application refers to the time at which half of the mosquitoes in each group die.

Example 1 isolation and identification of mosquito densovirus AalDV-6

The material and the method are as follows:

aedes albopictus densovirus AaldV-6(Aedes albopictus denovirus AaldV-6) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number: CCTCC V201652. The preservation date is as follows: year 2016, 11, 29, storage location: wuhan, Wuhan university, China.

0.01mM PBS phosphate buffer pH7.2 was purchased from Life Biotechnology, Inc., USA, the Viral genome Extraction Kit TaKaRa miniBEST Viral RNA/DNA Extraction Kit was purchased from Takara, Thermo Scientific Maxima Hot Start PCR Master Mix enzyme was purchased from ThermoFisher, T-vector was purchased from Promega, Trans1-T1 chemocompetent cells were purchased from Beijing Quanji Biotechnology, Inc., and both primer synthesis and DNA sequencing were performed by Shanghai Yijiki trade, Inc.

20 Aedes albopictus captured in Guangzhou field are taken as a group, the group is respectively ground by 2mL PBS buffer solution, the centrifugation is carried out for 15 minutes at 3750 r/min, the supernatant is collected, the filtration sterilization is carried out by a 0.22 mu m filter membrane, 100 mu L of the supernatant is taken to extract virus genome, primers are designed in an NS1 coding region conserved in mosquito densovirus, the mosquito densovirus is detected, the primers are designed by a Primer design tool Primer-blast of NCBI of American national institutes of health, and the Primer sequence is as follows:

reverse primer: 5'-GTTTCGATACCGTAACGGATGC-3'

Reaction procedure: pre-denaturation 95 ℃, annealing 55 ℃, extension 72 ℃ and extension time 15 seconds.

Carrying out electrophoresis detection and analysis on the agarose gel containing ethidium bromide, wherein the length of a PCR product is about 150bp, purifying and recovering the PCR product of a PCR detection positive sample, connecting a T vector, transforming Trans1-T1 chemical competent cells, and submitting the PCR product to sequencing of Shanghai Weijiji biotechnology Limited after colony PCR identification.

After PCR detection of each sample, a positive sample is obtained, a PCR product is connected with a T vector and then subjected to sequencing by a company, and blast is carried out on the PCR product and the densovirus found on NCBI, and the obtained result is similar to the found mosquito densovirus sequence.

EXAMPLE 2 proliferation of Aedes albopictus densovirus-6 in Aedes albopictus C6/36 cells

The material and the method are as follows:

the Viral genome extraction Kit TaKaRa miniBEST Viral RNA/DNAextraction Kit used in this experiment was purchased from Takara, EcoRV restriction enzyme was purchased from Thermo Fisher, SuperReal Premix Plus was purchased from Beijing Tiangen Biotech Ltd.

C6/36 cells were passaged to 25cm²And (3) removing the culture medium from the cell culture bottle, rinsing the cells once by using 1mL of PBS preheated at 28 ℃ after 24 hours, adding the AalDV-6 detected in the example 1, supplementing 1640 culture medium containing 5% fetal calf serum to 5mL, and culturing in a biochemical incubator at 28 ℃. 200 μ L of cell suspension was collected daily, stored at-80 ℃ and continued until day 7. The residual cell suspension was repeatedly frozen and thawed 3 times,after centrifugation at 3750 rpm for 10 minutes at 4 ℃, the supernatant was transferred to a new 15mL centrifuge tube for observation of virus morphology by electron microscopy as described in example 4.

After Dnase I removes DNA not packaged with virus particles from virus mixture due to MDV DNA virus, virus mixture was extracted with taka miniBEST Viral RNA/DNA virus genome extraction kit and eluted with 50 μ L of enzyme-free water, for details see the instructions. The concentration of the virus is detected by fluorescent quantitative PCR, and qPCR primers are as follows:

reverse primer: 5'-GTTTCGATACCGTAACGGATGC-3'

Preparing a fluorescent quantitative PCR reaction system:

Will carry outIn example 1, the PCR product-ligated T vector was used as a template, EcoRV was subjected to a single cleavage, the concentration of the recovered linear plasmid was measured, and the copy number per microliter of the linear plasmid in the recovered product was calculated according to the following formula (DNA mass × 6.022.022 6.022 × 10) (DNA mass 896.022 ×)²³) /(Length of template DNA × 1 × 10⁹× 660) and the DNA quality in the formula is the quality of the standard substance in qPCR, the length of the template DNA is the length of the standard substance, and the dilution by multiplying power is 10, and 5 points are selected as the standard substance for the absolute quantification of the virus to detect the total copy number of the virus in the virus liquid.

The reaction procedure was as follows:

the virus concentration was determined by the following formula, i.e., the virus concentration (one/ml) — copy number × 50 μ L/(200 μ L × 10)^- ³mL) was used, and 50. mu.L in the formula was the elution volume at the time of extracting the viral genome, and 200. mu.L was the volume of the viral suspension from which the genome was extracted, the results are shown in FIG. 1, and 1.34 × 10 from day 1 after AalDV-6 was added to C6/36⁹At the beginning of the individual/ml, the virus concentration showed a gradual increase, reaching a maximum of 9.32 × 10 by day 5⁹One cell/ml, which was 5.96-fold higher in virus concentration than day 1, indicates that AalDV-6 was able to proliferate in C6/36 cells after infecting C6/36 cells, and 5 days later, the cells began to age and the virus concentration began to decrease.

Example 3 proliferation of Aedes albopictus densovirus-6 in Aedes albopictus and in propagating water

The material and the method are as follows: TaKaRa miniBEST Viral RNA/DNA Extraction Kit, Superreal Premix Plus used in this experiment was purchased from Beijing Tiangen Biotech Ltd.

Dividing about 1000 molting Aedes albopictus first instar larvae into one group of 200 mols, adding into 2ml 10 ml of five groups¹⁰The AalDV-6 virus with the concentration per mL is used for detecting the concentration of the virus in the larvae of Aedes albopictus grown in and in the water body after 1, 3, 6, 9 and 12 days after infection. After 24 hours, dechlorinated water was added to 200mL of normal rearing. At the same time take outOne group of the water quality monitoring system collects 200 microliters of water for detecting the concentration of the viruses in the breeding water. All larvae were then triturated with 1mL PBS buffer, centrifuged at 3750 rpm for 15 minutes, the supernatant was sterile filtered through a 0.22 μm filter, 50 μ L of the extracted viral genome was removed and used to test the concentration of virus in the larvae, and the remaining virus was stored at-80 ℃. Samples were collected 1, 3, 6, 9, 12 days after infection as described above.

As shown in FIG. 2, it can be seen from FIG. 2 that, after AalDV-6 infection of 1 st larvae of Aedes albopictus, the viral infection started to invade and proliferate in larvae on day 1, 1.86 × 10¹⁰Start at one ml and reach a maximum of 1.65 × 10 by day 9¹²The virus concentration of the larvae per milliliter is 88.71 times higher, dead larvae and corpses are disintegrated in a water body or eaten by other larvae, so that the viruses in the larvae are released into the water, the virus concentration of the larvae in the water body is increased, and the larvae in the water body are 2.51 × 10 from day 1⁸Pieces/ml increased to day 9 at 3.65 × 10¹⁰The virus concentration is increased 145.42 times per ml, and the production of the first-instar larvae of the Aedes albopictus is obviously superior to the production of the virus by the C6/36 cell line of the Aedes albopictus in example 2. The virus has higher yield no matter in larva bodies or bred water bodies, wherein the larva bodies can be enriched to have higher virus content of 10¹²In units/ml.

Example 4 morphological identification of Aedes albopictus densovirus

The material and the method are as follows: the virus solution recovered in example 3 was centrifuged at 35000 rpm at 4 ℃ for 75 minutes to precipitate virus particles. The precipitated virus was further purified by centrifugation at 39000 rpm for 120 minutes at 4 ℃ in 1M sucrose buffer. Cesium chloride (0.3 g/ml) was added and centrifuged overnight at 60000 rpm at 8 ℃ to separate the virus by density gradient for DNA extraction and detection by electron microscopy. Purified virus particles were negatively stained with 2% PTA and photographed at 37000 magnification under the Philips CM12 electron microscope.

The results are shown in FIG. 3, and it can be seen from FIG. 3 that a large number of icosahedral virions with diameters of 22-25 nm can be observed under an electron microscope after separation and purification, and the surfaces of the virions are smooth; a small number of particles can be stained centrally with a negative staining agent, indicating that their nucleic acids have shed, empty viral capsids.

Example 5 Aedes albopictus densovirus-5 genomic sequence determination

The material and the method are as follows: the Viral genome extraction Kit TaKaRa miniBEST Viral RNA/DNAextraction Kit is purchased from Takara, T4 DNA library, PCR purification Kit, Klenow enzyme is purchased from Thermo fisher, Trans Stbl3 chemically competent cell is purchased from Beijing Quanjin Biotech Co., Ltd; DNA sequencing was conducted by Shanghai Weijie Jie based Biotech Ltd.

The genomic DNA of AalDV-6 was extracted from the TaKaRa miniBEST Viral RNA/DNA Extraction Kit. 10 units of klenow enzyme were added to the extracted AalDV-6 DNA, and the DNA was blunt-ended by incubation at room temperature for 15 minutes. The blunt-ended DNA was purified using a PCR purification kit. The cloning vector pKMV plasmid containing kanamycin resistance was digested with EcoRV restriction enzyme, and the linear plasmid was recovered by cutting gel. The blunt-ended AalDV-6 DNA and a linear plasmid pKMV are connected by T4 DNAlagase enzyme, the chemically competent Trans-Stbl3 is transformed, a single colony is picked for colony PCR identification, primers adopt M13F/M13R, and the sequence of the primers is as follows:

forward primer M13-F205 '-TGTAAAACGACGGCCAGT-3'

Reverse primer M13-Rev 5'-CAGGAAACAGCTATGACC-3'

Selecting positive clones, submitting to Shanghai Yiwei Jie based Biotechnology limited company for DNA sequencing, and performing bidirectional sequencing.

As a result: after colony PCR identification, positive clones were obtained and tested in both directions using M13-F20 and M13-Rev. The sequencing result is spliced by using a Seqman tool of DNAstar to obtain the complete sequence of AalDV-6 (see a sequence table 1):

the bioinformatics analysis of the sequence shows that the genome structure of the virus is similar to that of other Aedes aegypti and Aedes albopictus cell lines or separated from mosquito populations, and the virus has over 90 percent of homology, and is a new densovirus existing in mosquitoes. Encodes two non-structural proteins NS1 and NS2, and one structural protein VP, flanked by Inverted Terminal Repeat Sequences (ITRs) in which the AalDV-6 genome is 3981nt in full length (see fig. 4).

The strain is named as Aedes albopictus densovirus AalDV-6(Aedes albopictus denovirous sAalDV-6), is preserved in China center for type culture Collection with a preservation date of 2016, 11 and 29 days, and has a preservation number of CCTCC NO. V201652 and a preservation address of Wuhan university in China.

Example 6 Aedes albopictus densovirus-6 insecticide virulence test

The material and the method are as follows: AaldV-6 particles containing Aedes albopictus densovirus according to the method of example 3, AaldV-6 was added at 10⁸The concentration of the virus is infected by the first instar larvae of Aedes albopictus at one/ml concentration, the virus in the larvae of Aedes albopictus is separated and extracted after 7 days, and the concentration of AalDV-6 can be accurately determined according to a qPCR absolute quantitative method.

400 newly hatched first stage larvae were divided into 2 groups, aalDV-6 infected group and blank control group, respectively. Virus treatment group A suspension containing the virus was added to dechlorinated tap water so that AalDV-6 virus was 10¹⁰One/ml, while the control group was set, and kept for 24h without food. Feeding with turtle food, monitoring larva number until pupation, and placing pupae into mosquito cage. The larval, pupal and adult numbers were recorded daily and the experiment was terminated 28 days after infection. The probability unit method calculates the half lethal time LT50 of Aedes albopictus.

The results are shown in FIG. 5, from which it can be seen that Aedes albopictus was exposed to 10¹⁰The death peak started on day 7 with the individual/ml of the densovirus AalDV-6, and 28 days were observed, with the mortality rate of Aedes albopictus reaching 96.5%.The half lethal time of the densovirus AalDV-6 is calculated by a probability unit method from SPSS 22.0 to be 11.54 days, and the 95% confidence interval is 10.848-12.217.

From the foregoing detailed description of the invention, it will be apparent to those skilled in the art that modifications and variations of the present invention are possible, and that numerous modifications may be made in the methods and apparatus of the present invention without departing from the spirit and scope of the invention. Such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.

<110> under-illumination gold insurance

<120> Aedes albopictus densovirus and application thereof

<210>1

<211>3981

<212>DNA

<221> full sequence of Aedes albopictus densovirus AallDV-6

<400>1

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ctggaatcga aagaagattg gccaacgata accaacaacc agggctctca gatttatatt 421

gcaccgagac aatacatctt gcaactgcaa taccagaaag gagaaccatc gatcgagaaa 481

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ggtcacaggc aagcacgacc gaaatgctac gagacgctgt ccaaagatgc ggttttgaag 781

gccctcctaa cagcccaagc gaaaataaca gagatggaat tgatggaacg tgtatatcaa 841

ccgtggacat acaaagcaat tgtattgtta acgcacattg cccaaaacaa ggaccaagca 901

gtcaaaccaa taagagaaag aaatcaaccg atacaacaga atcaagcgga tccaaaaaaa 961

ataaaagcag caataatcaa caaaatatac aagaacaaag cagttccagc atcaccgacg 1021

cactcgatct cgtcgacgga gagttggatg gatcaactgg atcgaatcga gaaacagcat 1081

actacacatt cgtcctccac aaaaacaacg ttaaagagga ctggagatac atcgccacaa 1141

ccagggccaa gcaagcgccg agtttcatca cattcgatca cggagaccac atccatatcc 1201

tcttctcctc gtccaataca ggaggaaaca gcacaagagt cagaaccaga atcaccaagt 1261

ttcttagtgc aacaagcgca ggaagtgcag aagcgactat cactttttcc aaagttaaat 1321

ttctcaggaa ctacattctc tattgcatcc gttacggtat cgaaacagtc aatatctatg 1381

gaaataaaat ccaacaacaa ttaaccgaag caatggatac atttaaaata ttatttgaaa 1441

atagagaccc aaatgacgta atattagaag ccggatgcaa attatatcat gaagaaaaaa 1501

aggataataa acaaaaaaga tgcggacaac ggaagcaaca aaatctaacg gacattatat 1561

tggaaaaaat taaagaaaag aaaattacaa cggcacaaca atgggaaaat caaattgaac 1621

cggaattcaa aatacaatta atgaaagagt ttggattaaa tgtggacagt tatgtaacaa 1681

gaatagtacg catcgaaaga acacgtatac aacaattgat aaaagcaaaa acgcttacgg 1741

aaataatgct tgaaatatta aatgatgact atattaaaca cttcacacca ggagaagaca 1801

acagcaaaac aacaaaatgt attgaatgga tagaatatct attcaaagaa aataacatca 1861

atataatcca cttcttggca tggaatgaaa ttataaaaac aaaaagatat aaaaaaataa 1921

acggaatggt actagaaggt atcacaaacg caggaaaatc actaatatta gacaacttat 1981

tggccatggt taaaccagaa gaaataccac gagaacgaga caacagtgga ttccaccttg 2041

accaagtacc aggagcagga tcgatcctat ttgaagaacc aatgatcaca ccagtaaacg 2101

tcggaacatg gaaattatta ttagaaggaa aaaccataaa aacggatgta aaaaacaaag 2161

acaaggaacc gatagaacgc acaccaacgt ggatcacaac agcaactcca ataacaaaca 2221

acattgatat gaatgaaaca tcacaaatac tacaaagaat aaaattatat atattcaaaa 2281

aaagtatcca acacagagac gacaaatata ctataaatgc gcaaattcaa aataaattaa 2341

tcagtcgtcc tccaactctc attgagccaa tacatatggc catagtgttt ataaaaaatt 2401

tcacaaaaat atataatcta atcgcagaag aagacaaggc acacacagta aacgaaaagg 2461

caatacaaat aagcaacgaa gtgaaagaag aagcagaatc atggcagaca gcactacaat 2521

ggaccatgat ggagaacaac gaggaacaaa acgaaaacga gacgcaggag ctggaggatc 2581

aggtgctgga attggcaaag gagcaagcaa ctacgtaaaa gaaggatatg gaccaaatat 2641

gaccgaaatg gtaccaagaa acatattgaa taaaggaaat cacacagtct atcatgtggt 2701

aaaacaacaa aaatatctag acttcaatta cgtaacaaat caaaacccat acataattcc 2761

atatcaaaca gcaggattct gggcatcaat gtgggaccaa acagacatcg gatccaataa 2821

cagcattaat ataatgaaag cattaaacaa cgtatcacta ggagtaacat ggatcaaagg 2881

agaaatcaca ttcgaagtat attcagtaac aagacaacgc ttgctaacag gaacaacaaa 2941

ccaaactaca tgggactttg aaacaagtca aaacatgttc atcgcagatg cagacagaga 3001

accagaaaac ttcaacttag caacagcagc agcaactgga ccacttgcac aacaaacaac 3061

acaaacatta ctattcaatg caaacaatga cagatataca aaatatgaat taccacaaag 3121

aaaccaatat acaagggaaa ttaacttcca acaattaaca aacaactata tgtggaaacc 3181

attggacatc agcgctgcag caaactttag aagattgatc ccaatggcag aaggagtata 3241

tacaacatca aatgcaacaa gtaaaatgac agaattaaca aatcaaacta cagcatacgc 3301

cacatcaggc aaaacaacac aagcaacact attcagaaat agaacatcat accctagaat 3361

gcatatggca caaccacaag ttccagatga aaccggatac atgaaattca gataccaagt 3421

acgaatgagc acaaaactac atctcgaatt ccatctctac ccagattacg gcacatcaac 3481

aaacatagaa tatatgcaga gacaagtact ggaattacca gaagtaacag caacaggagg 3541

agtggtaaca tgtatgccgt atgaaatcaa aacttaaata ttaaattcaa cttgtatcaa 3601

ctataacaca tatataatca ataaagcatt caaaaaacat ataagtcaaa ttaatatata 3661

tcacaataaa aatccacctt aaaacataag cttaatttcc acctccgtat tccacctcag 3721

aatattggct taaaatccac ctccaatgat acagttagga agctaatatt agtccgggat 3781

ccccgtgtgg ccgataggcg aggatcgaaa gcccaaattt tgatgacgtc acctcacaca 3841

cataccaaaa gctttagttt ctaatagaaa cagcgtatta cgcttaaagc ttttggtatg 3901

tgtgtgaggt gacgtcatca a 3961。

Claims

1. An Aedes albopictus densovirus, which is characterized in that: the recombinant virus AalDV-6 is named as Aedes albopictus densovirus AalDV-6, is preserved in China center for type culture Collection, and has the preservation number of CCTCC number V201652, and the nucleotide of the Aedes albopictus densovirus AalDV-6 is shown in a sequence table SEQ ID NO. 1.

2. Use of the aedes albopictus densovirus of claim 1 for the preparation of a biological mosquito repellent.